This invention pertains to the extract of a species of Nerium, particularly Nerium Oleander, and to a method for production thereof by super critical carbon dioxide extraction.
Nerium oleander is an ornamental plant widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean. Its medical and toxicological properties have long been recognized. It has been used, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, and even in the induction of abortion. Oleandrin, an important component of oleander extract, is a potent inhibitor of human tumor cell growth. Oleandrin-mediated cell death is associated with calcium influx, release of cytochrome C from mitochondria, proteolytic processes of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation.
One extract component of the oleander plant is oleandrin. Oleandrin is a cardiac glycoside that is exogeneous and not normally present in the body. Oleandrin induces apoptosis in human but not in murine tumor cell lines (Pathak et al., Anti-Cancer Drugs, vol. 11, pp. 455-463, 2000), inhibits activation of NF-kB (Manna et al., Cancer Res., vol. 60, pp. 3838-3847, 2000), and mediates cell death through a calcium-mediated release of cytochrome C (McConkey et al., Cancer Res., vol. 60, pp. 3807-3812, 2000). A Phase I trial of an oleander extract has been completed recently (Mekhail et al., Am. Soc. Clin. Oncol., vol. 20, p. 82b, 2001). It was concluded that oleander extracts can be safely administered at doses up to 1.2 ml/m2/d. No dose limiting toxicities were found.
In addition to being selectively cytotoxic for tumor cells, cardiac glycosides may also enhance cell response to cytotoxic actions of ionizing radiation. Ouabain, a cardiac glycoside endogeneous to the body, was reported to enhance in vitro radiosensitivity of A549 human lung adenocarcinoma cells but was ineffective in modifying the radioresponse of normal human lung fibroblasts (Lawrence, Int. J. Radiat. Oncol. Biol. Phys., vol. 15, pp. 953-958, 1988). Ouabain was subsequently shown to radiosensitize human tumor cells of different histology types including squamous cell carcinoma and melanoma (Verheye-Dua et al., Strahlenther. Onkol., vol. 176, pp. 186-191, 2000). Although the mechanisms of ouabain-induced radiosensitization are still not fully explained, inhibition of repair from sublethal radiation damage and an increase in radiation-induced apoptosis have been advanced as possibilities (Lawrence, 2000; Verheye Dua et al., 2000; Verheye-Dua et al., Strahlenther. Onkol., vol. 172, pp. 156-161, 1996). The cardiac glycoside oleandrin also has the ability to enhance the sensitivity of cells to the cytotoxic action of ionizing radiation. See U.S. patent application Ser. No. 10/957,875 to Newman, et al.
Extraction of glycosides from plants of Nerium species has traditionally been carried out using boiling water. The process of using boiling water as an extraction method to obtain active ingredients from Nerium oleander yields many products. Among these are oleandrin, nerine, and other cardiac glycoside compounds. The plant extracts are useful in the treatment of cell-proliferative diseases in animals.
U.S. Pat. No. 5,135,745 to Ozel pertains to extracts of Nerium species and their use in the treatment of cell-proliferative diseases. Ozel obtains the extraction of Nerium oleander through the use of heat. In particular, sliced plant material is placed in distilled water and boiled until an appropriate density is reached. The mixture is then filtered and heated again.
U.S. Pat. No. 5,869,060 to Selvaraj et al. pertains to extracts of Nerium species and methods of production. To prepare the extract, plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterilized by filtration. The resultant extract can then be lyophilized to produce a powder.
Supercritical fluid extraction involves the use of a supercritical fluid to selectively extract a particular compound. A supercritical fluid is a liquid or a gas at atmospheric conditions, but becomes supercritical when it is compressed above its critical pressure and heated above its critical temperature. Supercritical fluids have increased dissolving power in their supercritical regions. A supercritical fluid exhibits properties between those of a gas and a liquid, and has the capacity to dissolve compounds that may only dissolve poorly or not at all in the gas or liquid state. Supercritical fluids are ideal for extraction of these compounds because they have high dissolving power at high densities and demonstrate good fractionation and separation of the compound from the fluid at lower densities when the pressure or temperature is changed. The general procedure of using supercritical carbon dioxide extraction in food processing industry has been described by Raventos, et al., in 2002 (M. Raventos, et al., Application and Possibilities of Supercritical CO2 Extraction in Food Processing Industry: An Overview, Food Sci Tech Int. Vol. 8 (5) (2002) 269-284), the entire content of which is hereby incorporated by reference.
It has been demonstrated that oleandrin is the principal cytotoxic component of Nerium oleander (Newman, et al., J. Herbal Pharmacotherapy, vol. 13, pp. 1-15, 2001). Methods to enhance the relative content of oleandrin from plant material are therefore warranted. While hot water extracts of Nerium oleander may provide oleandrin and related cardiac glycosides in relatively low yield, an improved method for obtaining a concentrated form of cardiac glycosides including oleandrin is needed.